CONSERVED GLU-181 AND ARG-182 RESIDUES OF ESCHERICHIA-COLI H-ATPASE (ATP SYNTHASE) BETA-SUBUNIT ARE ESSENTIAL FOR CATALYSIS - PROPERTIES OF33 MUTANTS BETWEEN BETA-GLU-181 AND BETA-LYS-801 RESIDUES()

Twenty-two mutants between beta Glu-161 and beta Lys-201 of Escherichi a coli H+-ATPase beta subunit could grow by oxidative phosphorylation, but 11 other such mutants, beta Glu-181-->Gln, Asp, Asn, Thr, Ser, Al e, or Lys and beta Arg-182-->Lys, Ala, Glu, or Gin, could not. The bet a Asp-181, beta Lys-182, and other defective mutants had 1.4, 1, and < 0.1%, respectively, of the wild-type membrane ATPase activity. Partia lly purified F-1-ATPases from all mutants at positions 181 and 182, ex cept for the beta Asp-181 and beta Lys-182 mutants, showed very low un isite catalysis. Purified F-1-ATPases of the beta Gln-181 and beta Ala -181 mutants showed no multisite (or steady state) catalysis and slow unisite catalysis (less than or equal to 1% of that of the wild type): their defects could be attributed to decreased catalytic rates (low k (+2) and k(-2)). Changes of the k(+2) and k(-2) values in the beta Asp -181 enzyme, which showed detectable multi- and unisite catalysis, wer e less marked (27 and 21%, respectively, of wild-type rates). The beta Gln-182 enzyme showed defective catalysis (less than or equal to 0.1% of the multi- and similar to 1% of the unisite catalyses of the wild type), whereas the beta Lys-182 enzyme showed 1 and 85% of the wild-ty pe multisite and unisite catalytic rates, respectively. beta Lys-182 h ad wild-type values of k(+2) and k(-2), but beta Gln-182 had k(+2) abo ut 10-fold lower than that of wild type. The position 181 and 182 muta nt enzymes had significantly increased K-d (k(-1)/k(+1)) values, refle cting decreased substrate binding. These results suggest that beta Glu -181 and beta Arg-182 are essential for substrate binding, although mu tations with conservative substitutions at these positions do not have drastic effects. This study also indicates the importance of the cons erved Gly-Glu-Arg (GER) sequence (beta 180-beta 182).